This R21 project seeks to develop new approaches for the genetic manipulation of mammalian oocytes, which we envision will accelerate advancing our knowledge of oocyte function and reproductive health. Genetic manipulation of mammalian oocytes has primarily used two methods: RNA interference and knockout mice. While RNAi has been a highly successful method of RNA ablation and subsequent protein knockdown in oocytes, RNAi approaches are not without limitations, as knockdown can be inefficient. Furthermore, double-stranded RNA and siRNAs have to be introduced into oocytes by microinjection, which is labor- and time-intensive and makes it impractical do use a large-scale RNAi approach (e.g., siRNA library-based screens). Knockout mice certainly have provided significant insights into mammalian oocyte biology as well, but knockout approaches also are not without pitfalls, including the time and expense involved in obtaining a knockout. This project seeks to develop alternatives to these methods, utilizing different established nucleic acid-based methods in novel combinations and with specialized modifications for the applications proposed here. In Aim 1, we will augment the use of siRNAs for post-transcriptional gene silencing with another reagent, a short single-stranded nucleic acid called a triplex-forming oligonucleotide (TFO) for pre-transcriptional silencing. TFOs bind to homopurine tracts in double-stranded DNA, and have been used to regulate gene expression in cultured cells and in vivo. The hypothesis for Aim 1 is that TFOs will inhibit transcription of a targeted gene, while siRNAs will mediate degradation of any residual mRNAs that were transcribed. This will be tested in vitro with isolated oocytes as well as with follicle-enclosed oocytes for longer-term culture. In Aim 2, we will develop methods for delivery of agents into oocytes. We will identify a novel agent for oocyte- specific delivery, using a screen of an aptamer library (with 1.2 X 1018 oligo-2'-deoxyribonucleotide sequence isomers) to isolate an aptamer that will interact with the oocyte's zona pellucida (ZP). Aptamers are nucleic acid-based molecules that bind with high affinity to target molecules. Aptamers can be used for delivery of agents such as siRNAs into cells; this delivery works in vivo, and aptamers currently are being developed as therapeutics to target drugs and other agents to specific cell types for treatment of a variety of diseases (13 aptamers are in clinical trials). Additionally, as an alternative tool, we will also test a cell-penetrating peptide for intra-oocyte delivery. We will couple siRNAs or TFOs to ZP-binding aptamers and/or a cell-penetrating peptide, and test these for their actions in oocytes. The future direction of this work will be to test the ZP-targeting aptamer for systemic delivery of siRNAs and TFOs, as a means of in vivo oocyte-specific knockdown as an alternative to knockout/transgenic methodologies, as well as potentially the foundation of a novel female contraceptive.